My long-range goal is to assess and improve the capability of diffuse optical methods in detection, diagnosis and therapeutic monitoring of breast cancer. Diffuse optical methods using near-infrared light provide several unique functional parameters (such as total hemoglobin concentration, blood oxygen saturation, blood flow, water and lipid concentrations) to enhance tumor sensitivity and specificity. Moreover, deep tissue micro-environment (e.g. vascular leakiness, glucose metabolism) can be probed with diffuse fluorescence imaging/spectroscopy. Diffuse optical methods can impact cancer therapy monitoring and planning at early stages since the metabolic changes due to therapy may precede the anatomical size changes. During the mentored K99 phase, I explored the utility of portable diffuse optical device to assess neoadjuvant chemotherapy response of human subjects with breast cancer, and received trainings in tumor biology, biostatistics and biomedical imaging. The proposed research builds on initial success from K99 phase and aims to expand the monitoring capability of dffuse optics by incorporating fluorescence detection. Using new targeted optical fluorescent dyes, one can probe tumor specific metabolism and physiology not accessible by endogenous contrast alone. In particular, I will explore deoxyglucose analogue fluorescent dye to test its ability to probe glucose metabolism, which has been successfully utilized in PET imaging for therapy monitoring and diagnosis. First, I will build preclinical study platform consisting of animal tumor models and diffuse optical imaging/spectroscopy systems to quantify both endogenous and exogenous tumor contrast accurately. The fluorescent dye will be characterized for its biodistribution, pharmacokinetics, and tumor-to-normal contrast in mouse xenograft model by microscopic and macroscopic fluorescence imaging techniques, and will be compared to autoradiography and PET. Then, I will investigate whether simultaneous monitoring of glucose and oxygen metabolism accessible by all optical methods can enhance the assessment of treatment efficacy. This research will pave the ways for molecular imaging in vivo for therapy monitoring when it becomes available for human use.